This invention relates to the disease atherosclerosis, methods of modulating the formation of atherosclerotic lesions, and methods of identifying compounds which modulate atherosclerotic lesion formation. Specifically the invention relates to the reduction of atherosclerosis through the modulation of LDL-proteoglycan binding at Site B (amino acids 3359-3369) of the apo-B100 protein in LDL.
High levels of LDL are a major risk factor for coronary disease and are the source for most of the cholesterol that accumulates in the arterial wall (Ross, R. 1995. Annu. Rev. Physiol. 57:791-804). Subendothelial retention of LDL has been suggested to be a key pathogenic process in atherosclerosis, and several lines of circumstantial evidence suggest that intramural retention of atherogenic lipoproteins involves the extracellular matrix, chiefly proteoglycans (Hurt-Camejo, E. et al. 1997. Arterioscler Thromb Vasc Biol. 17:1011-1017; Williams, K. J., and I. Tabas. 1995. Arterioscier. Thromb. Vasc. Biol. 15:551-561; and Radhakrishnamurthy, B. et al. 1990. Eur. Heart J. 11 Suppl E: 148-157).
The significance of the possible LDL proteoglycan interaction has been highlighted in two recent review articles (Hurt-Camejo, E. et al. 1997. Arterioscler Thromb Vasc Biol. 17:1011-1017; and Williams, K. J., and I. Tabas. 1995. Arterioscier. Thromb. Vasc Biol. 15:551-561). Williams and Tabas proposed that subendothelial retention of atherogenic lipoproteins is the central pathogenic process in atherosclerosis. Moreover, they hypothesized that retained lipoproteins can directly or indirectly provoke all known features of early lesions, such as lipoprotein oxidation, monocyte migration into the artery wall, macrophage foam cell formation, and cytokine production, and can accelerate further retention by stimulating local synthesis of proteoglycans. Several lines of evidence indicate that the retention of arterial lipoproteins involves the extracellular matrix; proteoglycans in particular have been hypothesized to play an important role (Hurt-Camejo, E. et al. 1997. Arterioscler Thromb Vasc Biol. 17:1011-1017; Williams, K. J., and I. Tabas. 1995. Arterioscier. Thromb. Vasc Biol. 15:551-561; Camejo, G. et al. 1988. Arteriosclerosis. 8:368-377; and Hurt, E., and G. Camejo. 1987. Atherosclerosis. 67:115-126). First, purified arterial proteoglycans, especially those from lesion-prone sites (Cardoso, L. E., and P. A. Mourao. 1994. Arterioscler. Thromb. 14:115-124; and Ismail, N. et al. 1994. Atherosclerosis. 105:79-87), bind atherogenic lipoproteins in vitro, particularly LDL from patients with coronary artery disease (Lindxc3xa9n, T. et al. 1989. Eur. J Clin. Invest. 19:38-44). LDL binds with high affinity to dermatan sulfate and chondroitin sulfate proteoglycans produced by proliferating smooth muscle cells (Camejo, G. et al. 1993. J. Biol Chem. 268:1413-1437). Second, proteoglycans are a major component of the artery wall extracellular matrix and are available to participate in the interactions of lipoproteins in the earliest stages of atherogenesis. Third, retained apo-B immunologically co-localizes with proteoglycans in early and developed lesions (Walton, K., and N. Williamson. 1968. J. Atheroscler. Res. 8:599-624; Hoff, H., and G. Bond. 1983. Artery. 12:104-116; Hoff, H. F., and W. D. Wagner. 1986. Atherosclerosis. 61:231-236; Nievelstein-Post, P. et al. 1994. Arterioscler. Thromb. 14:1151-1161; and Galis, Z. et al. 1993. Am J. Pathol. 142:1432-1438). The observation that the arterial wall content of these proteoglycans increases during atherosclerosis and correlates with an increased accumulation of aortic cholesterol also supports the potential importance of the interaction between LDL and proteoglycans (Hoff, H. F., and W. D. Wagner. 1986. Atherosclerosis. 61:231-236; Merrilees, M. et al. 1990. Arteriosclerosis. 81:245-254).
Proteoglycans contain long carbohydrate side-chains of glycosaminoglycans, which are covalently attached to a core protein by a glycosidic linkage. The glycosaminoglycans consist of repeating disaccharide units, all bearing negatively charged groups, usually sulfate or carbohydrate groups. In vitro, LDL bind with high affinity to many proteoglycans found in the artery wall, including dermatan sulfate proteoglycans (e.g., biglycan) and chondroitin sulfate proteoglycans (e.g., versican), which are produced by smooth muscle cells in response to PDGF or TGFxcex2 (Schonherr, E. et al. 1991. J. Biol. Chem. 266:17640-17647; and Schxc3x6nherr, E. et al. 1993. Arterioscler. Thromb. 13:1026-1036). The interaction between LDL and proteoglycans have been hypothesized to involve clusters of basic amino acids in apo-B100, the protein moiety of LDL, that interact with the negatively charged glycosaminoglycan proteoglycans (Mahley, R. et al. 1979. Biochem. Biophys. Acta. 575:81-91; Carnejo, G. et al. 1988. Arteriosclerosis. 8:368-377; Weisgraber, K., and S. Rall, Jr. 1987. J. Biol. Chem. 262:11097-11103; and Hirose, N. et al. 1987. Biochemistry. 26:5505-5512) or by bridging molecules such as apo-E or lipoprotein lipase (Williams, K. J., and I. Tabas. 1995. Arterioscier. Thromb. Vasc. Biol. 15:551-561).
Isolation of large fragments of apo-B100 from different regions characterized by concentrations of positive clusters indicated that up to eight specific regions in apo-B100 bind proteoglycans (Camejo, G. et al. 1988. Arteriosclerosis. 8:368-377; Weisgraber, K., and S. Rall, Jr. 1987. J. Biol. Chem. 262:11097-11103; and Hirose, N. et al. 1987. Biochemistry. 26:5505-5512). Weisgraber, K., and S. Rall, Jr. 1987. J. Biol. Chem. 262:11097-11103 identified two fragments, residues 3134-3209 and 3356-3489, that bind to heparin with the highest affinity. Recently Camejo and coworkers confirmed this finding and proposed that residues 3147-3157 and 3359-3367 may act cooperatively in the association with proteoglycans (Hurt-Camejo, E. et al. 1997. Arterioscler Thromb Vasc Biol. 17:1011-1017; and Olsson, U. et al. 1997. Arterioscler. Throm. Vasc. Biol. 17:149-155). However, because these studies were carried out with delipidated apo-B fragments in the presence of urea or with short synthetic apo-B peptides, it is not clear which of the binding sites are functionally expressed on the surface of LDL particles. Some or many of these postulated glycosaminoglycan-binding sites may not be functional when apo-B is associated with LDL. For example, apo-E has two heparin-binding sites, but only one binds to heparin when apo-E is completed with phospholipid (Weisgraber, K. et al. 1986. J. Biol Chen 261:2068-2076). This heparin-binding site coincides with the LDL receptor-binding site of apo-E.
Although eight potential glycosaminoglycan-binding sites have been identified in apo-B100 (Camejo, G. et al. 1988. Arteriosclerosis. 8:368-377; Weisgraber, K., and S. Rall, Jr. 1987. J. Biol. Chem. 262:11097-11103; and Hirose, N. et al. 1987. Biochemistry. 26:5505-5512), it was not known which of them participate in the physiological binding of LDL to proteoglycans. Previously, we have demonstrated, in conjunction with others, that Site B (residues 3359-3369) is the LDL receptor-binding site, and in the study which generated the present invention we found that it is also the primary binding site to proteoglycans.
Modification of LDL potentially exposes the other proteoglycan-binding sites. Paananen and Kovanen (Paananen, K., and P. T. Kovanene. 1994. J. Biol. Chem. 269:2023-2031) noted that proteolysis of apo-B100 strengthened the binding of LDL to proteoglycans, suggesting the exposure of buried heparin binding sites. Likewise, when LDL are fused by sphingomyelinase treatment, the modified lipoproteins bind more avidly to proteoglycans. The finding that multiple heparin molecules bind to LDL (Cardin, A. et al. 1987. Biochemistry. 26:5513-5518) may also be explained by a cooperative effect of heparin binding to one site that triggers a conformational change in apo-B100 that enables other sites to participate in the interaction. Thus, the initial interaction with proteoglycans may induce structural alterations of the LDL that expose heparin/proteoglycan-binding sites that may contribute to the intramural retention of LDL after the initial interaction with the primary binding site.
The interaction between LDL and the LDL receptor plays a major role in determining plasma cholesterol levels in humans and other mammalian species (Goldstein, J. et al. 1985. Annu. Rev. Cell Biol. 1:1-39). Apo-B100 is the major protein component of LDL and is responsible for the binding of these lipoproteins to the LDL receptor (Innerarity, T. et al. 1990. J. Lipid Res. 31:1337-1349). The relevance of this catabolic pathway is best illustrated by the genetic disorders familial hypercholesterolemia (FH) and familial defective apo-B100 (FDB), in which high levels of LDL accumulate in the circulation because mutations in the LDL receptor (FH) or in the ligand (FDB) disrupt the binding of LDL to its receptor (Innerarity, T. et al. 1990. J. Lipid Res. 31:1337-1349). Many different mutations of the LDL receptor cause FH (Hobbs, H. et al. 1992. Hum. Mutat. 1:445-466), but FDB is associated with a single site mutation, the substitution of glutamine (Innerarity, T. et al. 1987. Proc. Natl. Acad. Sci. USA. 84:6919-6923) or, in a few cases, tryptophan (Gaffney, D. et al. 1995. Arterioscler. Thromb. Vasc. Biol. 15:1025-1029) for the normally occurring arginine at residue 3500 of apo-B100. With the exception of an arginine-3531 to cysteine mutation (Pullinger, C. et al. 1995. J. Clin. Invest. 95:1225-1234), which is associated with a minor decrease in LDL receptor binding, extensive searches have not found any other mutations of apo-B100 that cause defective receptor binding of LDL (Pullinger, C. et al. 1995. J. Clin. Invest. 95:1225-1234). The FDB mutation occurs at an estimated frequency of 1/500 in the normal population and is therefore one of the most common known single-gene defects causing an inherited abnormality (Innerarity, T. et al. 1990. J. Lipid Res. 31:1337-1349).
Much attention has focused on understanding the molecular interaction between apo-B100 and the LDL receptor. The structural and functional domains of the LDL receptor have been defined in detail (Hobbs, H. et al. 1992. Hum. Mutat. 1:445-466), but much less is understood about the receptor-binding domain of apo-B100, because of its large size and insolubility in aqueous buffer. Furthermore, both the lipid composition and the conformation of apo-B100 appear to be crucial to its function as an effective ligand for the LDL receptor, since apo-B100 binds to the LDL receptor only after the conversion of large VLDL to smaller LDL (Goldstein, J. et al. 1985. Annu. Rev. Cell Biol. 1:1-39).
Selective chemical modification of the apo-B100 of LDL demonstrated that the basic amino acids arginine and lysine were important in the interaction of LDL with its receptor (Mahley, R. et al. 1977. J. Biol. Chem. 252:7279-7287; and Weisgraber, K. et al. 1978. J. Biol. Chem. 253:9053-9062). Once apo-B100 was sequenced, several regions enriched in arginine and lysine residues became candidates for receptor binding, including Site A (residues 3147-3157) and Site B (residues 3359-3367) (Knott, T. et al. 1985. Science. 230:37-43).
While it had been hypothesized that LDL-proteoglycan binding was possibly important to the formation of atherosclerotic lesions through the retention of lipoproteins in the subendothelium, this hypothesis has not been empirically demonstrated in the art. Moreover, there have been six obstacles which have prevented other researchers from demonstrating the mechanism by which atherogenesis occurs and using this information to combat atherosclerosis. First, there have been eight potential sites identified in the apo-B100 protein, any one or several of which could have been responsible for proteoglycans trapping LDL in the subendothelium. Second, it has been unknown which potential sites in the apo-B100 are exposed to the surface of the LDL particles and which are buried within the lipid core. Third, there has been evidence that some of the potential proteoglycan binding sites on apo-B100 may work cooperatively, creating the possibility that blocking proteoglycan binding at any single site might not have proven both necessary and sufficient to eliminate LDL retention in the subendothelium. Fourth, the modification of LDL has been shown in some cases to expose new proteoglycan binding sites to the surface. Fifth, any disruption to LDL proteoglycan binding had the potential to disrupt LDL receptor binding, which would serve to disrupt the natural clearance of LDL from blood, raise serum cholesterol levels, and potentially result in a condition similar to familial hypercholesterolemia. Sixth, it has not been possible to use site-directed mutagenesis and express the entire mutated apo-B100 proteins as LDL in order to define the proteoglycan-binding sites on LDL.
We have discovered that the amino acids of Site B in the apo-B100 protein are responsible for conferring proteoglycan binding activity on LDL. Recombinant LDL in which lysine3363 in apo-B100 was changed to glutamic acid has severely defective proteoglycan binding activity but normal LDL receptor-binding activity. Thus, the proteoglycan-binding and the receptor-binding activities in LDL can be separated by the introduction of a single point mutations into the apo-B100 protein, indicating that pharmaceutical strategies for disrupting LDL-proteoglycan binding need not inhibit LDL receptor binding.
Moreover, we have demonstrated for the first time in vivo that LDL-proteoglycan binding is necessary to the formation of atherosclerotic lesions and the onset of atherosclerosis. Transgenic mice expressing the mutant RK3359-3369SA apo-B100 LDL, which is defective for proteoglycan binding, was found to have strikingly less atherosclerosis than mice expressing the wild-type recombinant LDL, when both were fed a high cholesterol diet. These results demonstrate that disruption of LDL-proteoglycan binding at Site B in the apo-B100 protein is a credible target for pharmaceutical intervention for the reduction and elimination of atherosclerosis.
The present invention relates to the prevention of atherosclerosis through the modulation of LDL-proteoglycan binding at Site B (amino acids 3359-3369) of the apo-B100 protein in LDL. The invention encompasses apo-B100 proteins with mutations in Site B and which exhibit reduced binding to proteoglycans, fragments of these proteins containing Site B, and LDL particles comprising such mutants. The invention includes purified apo-B100 proteins comprising a mutation in Site B which results in reduced LDL-proteoglycan binding activity while maintaining LDL/LDL receptor binding (proteoglycanxe2x88x92receptor+mutant), including, for example, the K3363E mutation. The inventions also includes polypeptide fragments of these proteins which comprise the amino acid sequence of Site B in the apo-B100 protein of the invention, wherein said Site B is flanked on at least one side by a contiguous sequence of amino acids which is directly adjacent to Site B in the wild-type human apo-B100 sequence. The invention encompasses LDL particles and other lipoproteins which comprise an apo-B100 protein or protein fragment of the invention.
Accordingly, in certain embodiments, the invention provides mutant apo-B100 proteins and mutant apo-B100 polypeptide fragments, as well as LDL particles and other lipoproteins comprising a mutant apo-B100 protein or polypeptide fragment, which comprise a mutant Site B selected from one of the following Site B sequences:
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Glu3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:1);
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Asp3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:2);
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Ala3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:3);
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Thr3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:4);
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Ser3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:5);
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Gly3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:6);
Thr3358-Arg3359-Leu3360-Thr3361-Gly3362-Lys3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:7);
Thr3358-Arg3359-Leu3360-Thr3361-Asp3362-Lys3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:8);
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-LYS3363-Glu3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:9);
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Lys3363-Asp3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:10);
Thr3358-Glu3359-Leu3360-Thr3361-Arg3362-Lys3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:11); and
Thr3358-Asp3359-Leu3360-Thr3361-Arg3362-Lys3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:12); as well as Site B sequences with deletions, such as:
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:13);
Thr3358-Arg3359-Leu3360-Thr3361-Lys3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:14); and
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Lys3363-Gly3365-Leu3366-Lys3367(SEQ ID NO:15); and Site B sequences which include insertions, such as:
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Glu-Lys3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:16);
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Lys3363-Glu-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:17);
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Asp-Lys3363-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:18); and
Thr3358-Arg3359-Leu3360-Thr3361-Arg3362-Lys3363-Asp-Arg3364-Gly3365-Leu3366-Lys3367(SEQ ID NO:19).
The invention also includes antibodies which bind to antigenic determinants comprising Site B of the mutant apo-B100 proteins of the invention, including antibody compositions which bind to an antigenic determinant in an apo-B100 protein or protein fragment of the invention, wherein said antigenic determinant is not present in the wild-type human apo-B100 protein.
The invention also encompasses polynucleotides encoding the mutant apo-B100 proteins of the invention, targeting vectors and methods for creating mutant apo-B100 genes of the invention. The invention includes polynucleotides which encode an apo-B 100 protein or protein fragment of the invention, as well as cells comprising a polynucleotide of the invention or expressing an apo-B100 protein or protein fragment of the invention. The invention also includes non-human animals and mammals which comprise a polynucleotide of the invention or express an LDL, apo-B100 protein, or protein fragment of the invention.
The invention encompasses methods for preventing or reducing the severity of atherosclerosis in an animal or mammal, comprising the step of expressing a polynucleotide, LDL, apo-B100 protein, or protein fragment of the invention. Normally, a polynucleotide encoding an apo-B100 protein or protein fragment of the invention is transduced into a cell. The cell may be transduced ex vivo, then transferred into the animal or mammal, or the cell may be transduced in situ.
The present invention further encompasses methods of screening for and identifying inhibitors of LDL-proteoglycan binding, including drug screening assays based on simple LDL-proteoglycan binding, high through-put drug screening assays based on LDL-proteoglycan binding, two step LDL/proteoglycan and LDL/LDL-receptor binding assays, and in transgenic animals which express recombinant LDL.
The present invention encompasses methods for identifying inhibitors of LDL-proteoglycan binding, comprising the steps of:
(a) incubating a mixture comprising (i) proteoglycan, (ii) LDL, and (iii) a candidate compound, under conditions wherein LDL binds to proteoglycan to form an LDL-proteoglycan complex in the absence of said candidate compound;
(b) determining any difference between the amount of LDL-proteoglycan complex present in:
(i) the mixture prepared in step (a), and
(ii) a control mixture comprising said proteoglycan and said LDL in the absence of said candidate compound; and optionally
(c) correlating any difference determined in step (b) with said candidate compound""s ability to affect LDL-proteoglycan binding.
The present invention also encompasses identifying compounds which affect LDL-proteoglycan binding, which do not substantially affect LDL receptor binding, which further comprising the steps of:
(d) incubating a mixture comprising (i) LDL receptor, (ii) LDL, and (iii) a candidate compound that affects LDL-proteoglycan binding identified in step (c), under conditions wherein LDL binds to LDL receptor to form an LDL-LDL receptor complex in the absence of said inhibitor of LDL-proteoglycan binding;
(e) determining any difference between the amount of LDL-LDL receptor complex present in:
(i) the mixture prepared in step (d), and
(ii) a control mixture comprising said LDL receptor and said LDL in the absence of said inhibitor of LDL-proteoglycan binding; and optionally
(f) correlating any difference determined in step (e) with the LDL-LDL receptor binding activity of said candidate compound that affects LDL-proteoglycan binding.
In accordance with the instant invention, either the LDL or the proteoglycan of step (a) may be adhered to a solid support. Additionally, where the LDL is adhered to a solid support, the proteoglycan may be labeled, or where the proteoglycan is adhered to a solid support, the LDL may be labeled.
The invention further encompasses methods for identifying compounds which modulate atherosclerosis and/ LDL-proteoglycan binding in vivo, comprising the steps of:
(a) administering a candidate compound to a transgenic non-human animal which expresses a human apo-B gene, under conditions wherein measurable atherosclerotic lesions form in the arteries of said animal in the absence of said candidate compound;
(b) determining any difference between the extent of atherosclerosis present in:
(i) the animal of step (a), and
(ii) a control transgenic non-human animal in the absence of said candidate compound; and optionally
(c) correlating any difference determined in step (b) with the said candidate compound""s ability to modulate atherosclerosis in vivo.
The present invention further encompasses the compounds identified by the screening methods of the invention, including the compounds which affect, modulate, stimulate or inhibit of LDL-proteoglycan binding identified by the methods for identifying compounds that affect LDL-proteoglycan binding, as well as the compounds that affect, modulate, stimulate, or inhibit LDL-proteoglycan binding, which do not substantially affect LDL receptor binding identified by the methods for identifying inhibitors of LDL-proteoglycan binding, which do not eliminate LDL receptor binding, and the compounds which modulate, stimulate, or inhibit atherosclerosis in vivo identified by the methods for identifying compounds which modulate atherosclerosis in vivo. In addition the invention encompasses methods of inhibiting atherosclerosis in a human comprising administering to the human an agent that inhibits LDL-proteoglycan binding, or any of the other compounds identified by the methods of the invention.